Antioxidant effects of resveratrol and other stilbene derivatives on oxidative stress and *NO bioavailability: Potential benefits to cardiovascular diseases

Oxidative stress plays an important part in the appearance and development of cardiovascular diseases. In this context, overproduction of reactive oxygen species leads to deregulation of metabolic pathways, such as cell proliferation or inflammation, which interferes with the homeostasis of vascular endothelium. Oxidative stress can decrease the bioavailability of nitric oxide (*NO) in vessels. This decrease is highly associated with endothelial dysfunction. The "French paradox" is a phenomenon that associates a diet rich in saturated fatty acids and a moderate consumption of wine to a low prevalence of cardiovascular diseases. During the past 10 years, the beneficial effects of wine on cardiovascular diseases have been attributed to the actions of resveratrol and other polyphenols. One of the mechanisms involved in these beneficial effects is the capacity of resveratrol and some other stilbene derivatives to maintain sufficient *NO bioavailability in vascular endothelium. This review presents the latest findings on the molecular effects of resveratrol and other stilbene derivatives on the various actors that modulate *NO bioavailability during oxidative stress.     

The influence of wine polyphenols on reactive oxygen and nitrogen species production by murine macrophages RAW 264.7

The aim was to study the antioxidant properties of four wine polyphenols (flavonoids catechin, epicatechin, and quercetin, and hydroxystilbene resveratrol). All three flavonoids exerted significant and dose-dependent scavenging effects against peroxyl radical and nitric oxide in chemical systems. The scavenging effect of resveratrol was significantly lower. All polyphenols decreased production of reactive oxygen species (ROS) by RAW264.7 macrophages. Only quercetin quenched ROS produced by lipopolysaccharide-stimulated RAW264.7 macrophages incubated for 24 h with polyphenols. Quercetin and resveratrol decreased the release of nitric oxide by these cells in a dose-dependent manner which corresponded to a decrease in iNOS expression in the case of quercetin. In conclusion, the higher number of hydroxyl substituents is an important structural feature of flavonoids in respect to their scavenging activity against ROS and nitric oxide, while C-2,3 double bond (present in quercetin and resveratrol) might be important for inhibition of ROS and nitric oxide production by RAW 264.7 macrophages.     

Beneficial effects of Provinols: cardiovascular system and kidney

Red wine polyphenols have been reported to exert beneficial effects in preventing cardiovascular diseases but their molecular mechanisms of hemodynamic effects on functional cardiovascular and renal changes were studied much less. The review is focused on in vitro as well as in vivo effects of red wine extract containing polyphenolic compounds (Provinols) on cardiovascular systems and kidney in relation to the molecular and biochemical mechanisms of these compounds. This review provides the evidence that Provinols is able to produce ex vivo endothelium-dependent relaxation as a result of enhanced NO synthesis. Administration of Provinols partially prevents the development of hypertension during NO deficiency and accelerates the decrease of blood pressure in already established hypertension. The effects of Provinols include prevention and/or attenuation of myocardial fibrosis, reduction of aortic wall thickening and improvement of vascular functions. These functional and structural alterations are associated with significant augmentation of NO production, seen as the increase of NO synthase activity and eNOS protein expression. Moreover, it has been documented that Provinols decreased the oxidative stress within the cardiovascular system and kidney.     

Cardiovascular effects and molecular targets of resveratrol

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenol phytoalexin present in a variety of plant species and has been implicated to explain the health benefits of red wine. A wide range of health beneficial effects have been demonstrated for resveratrol in animal studies. In this review, we summarize the cardiovascular effects of resveratrol with emphasis on the molecular targets of the compound. In this regard, resveratrol stimulates endothelial production of nitric oxide, reduces oxidative stress, inhibits vascular inflammation and prevents platelet aggregation. In animal models of cardiovascular disease, resveratrol protects the heart from ischemia-reperfusion injury, reduces blood pressure and cardiac hypertrophy in hypertensive animals, and slows the progression of atherosclerosis. A number of direct and indirect target molecules mediating the aforementioned cardiovascular effects of resveratrol have been identified. These include, among others, the estrogen receptor α, the adenosine receptors, the cyclooxygenase 1, the histone/protein deacetylase sirtuin 1, the AMP-activated protein kinase, the Akt kinase, the nuclear factor-E2-related factor-2, and NF-κB. Molecular mechanisms involved in the signal cascades are discussed.     

Longevity nutrients resveratrol, wines and grapes

A mild-to-moderate wine drinking has been linked with reduced cardiovascular, cerebrovascular, and peripheral vascular risk as well as reduced risk due to cancer. The reduced risk of cardiovascular disease associated with wine drinking is popularly known as French Paradox. A large number of reports exist in the literature indicating that resveratrol present in wine is primarily responsible for the cardioprotection associated with wine. Recently, resveratrol was shown to extend life span in yeast through the activation of longevity gene SirT1, which is also responsible for the longevity mediated by calorie restriction. This review summarizes the reports available on the functional and molecular biological aspects of resveratrol, wine and grapes in potentiating the longevity genes.     

Antiangiogenic properties of natural polyphenols from red wine and green tea

Epidemiological studies have indicated that regular consumption of red wine and green tea is associated with a reduced risk of coronary heart disease and tumor progression. The development of tumors and of atherosclerosis lesions to advanced plaques, which are prone to rupture, is accelerated by the formation of new blood vessels. These new blood vessels provide oxygen and nutrients to neighboring cells. Therefore, recent studies have examined whether red wine polyphenolic compounds (RWPCs) and green tea polyphenols (GTPs) have antiangiogenic properties. In vitro investigations have indicated that RWPCs and GTPs are able to inhibit several key events of the angiogenic process such as proliferation and migration of endothelial cells and vascular smooth muscle cells and the expression of two major proangiogenic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2, by both redox-sensitive and redox-insensitive mechanisms. Antiangiogenic properties of polyphenols have also been observed in the chick embryo chorioallantoic membrane since the local application of RWPCs and GTPs strongly inhibited the formation of new blood vessels. Moreover, intake of resveratrol or green tea has been shown to reduce corneal neovascularization induced by proangiogenic factors such as VEGF and fibroblast growth factor in mice. The ability of RWPCs and GTPs to prevent the formation of new blood vessels contributes, at least in part, to explain their beneficial effect on coronary heart disease and cancer. This review focuses on the antiangiogenic properties of natural polyphenols and examines underlying mechanisms.     

Modulation of endothelial nitric oxide by plant-derived products

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is recognised as a central anti-inflammatory and anti-atherogenic principle in the vasculature. Decreased availability of NO in the vasculature promotes the progression of cardiovascular diseases. Epidemiological and clinical studies have demonstrated that a growing list of natural products, as components of the daily diet or phytomedical preparations, may improve vascular function by enhancing NO bioavailability. In this article we first outline common pathways modulating endothelial NO production or bioavailability to provide a basis for subsequent mechanistic discussions. Then we comprehensively review natural products and plant extracts known to positively influence eNOS activity and/or endothelial function in vitro or in vivo.We will discuss red wine, highlighting polyphenols, oligomeric procyanidins (OPC) and resveratrol as modulators of endothelial NO production. Other dietary products and their active components known to activate eNOS include cocoa (OPC and its monomer (?)-epicatechin), pomegranates (polyphenols), black and green tea (flavanoids, especially epigallocatechin gallate), olive oil (oleic acid and polyphenols), soy (genistein), and quercetin, one of the most abundant flavonoids in plants. In addition, phytomedical preparations made from ginkgo, hawthorn and ginseng, as well as formulations used in traditional Chinese Medicine, have been shown to affect endothelial NO production. Recurring phytochemical patterns among active fractions and purified compounds are discussed.In summary, there is increasing evidence that several single natural products and plant extracts influence endothelial NO production. Identification of such compounds and characterisation of their cellular actions may increase our knowledge of the regulation of endothelial NO production and could provide valuable clues for the prevention or treatment of cardiovascular diseases.     

Red wine polyphenols prevent endothelial damage induced by CCl4 administration

It became evident in the present study that carbon tetrachloride (CCl(4)), in addition to its known liver and renal toxicity, causes serious damage to endothelial cells. The preventive effect of red wine on cardiovascular diseases has been documented in a number of human population studies as well as in animal experimental models. In this study, the endothelium protective effect of polyphenolic compounds isolated from red wine was studied in rats administered 0.5 ml of CC(4)/kg body weight intraperitoneally twice a week for 8 weeks. Endothelemia (endothelial cells/10 microl of plasma) was used as the marker of endothelial cell injury in vivo. Chronic CCl(4) treatment for 8 weeks lead to a 3-fold increase of free endothelial cells circulating in the blood when compared to the baseline values (2.5+/-0.3). Parallel oral administration of polyphenols 40 mg/kg/day significantly decreased the endothelemia. Polyphenolic compounds alone did not produce significant changes. Three weeks of spontaneous recovery after the 8-week treatment with CCl(4) did not lead to a marked decrease of endothelemia, but the administration of red wine polyphenols during the 3-week period significantly decreased free endothelial cells in the blood. It can be concluded that long-term administration of CCl(4) may serve as a useful experimental model of endothelial damage. The red wine polyphenolic compounds exert a powerful protective effect on endothelial cells from the injury caused by CCl(4). This effect was documented by decreased endothelemia that corresponded to diminished endothelial cell swelling and detachment evaluated by histology of the vascular intima. The endothelium protective effect may be one of the key factors that contribute to the preventive action of red wine on cardiovascular diseases.     

The "French Paradox" and beyond: neuroprotective effects of polyphenols

Chronic ethanol ingestion is known to cause oxidative damage to a number of organs including the brain. This is partly due to the ability of ethanol to enhance oxygen free radical production and lipid peroxidation. Increase in oxidative stress has been regarded as an important underlying factor for a number of human health problems including cardiovascular diseases, aging, as well as many age-related neurodegenerative diseases. The strikingly low incidences of coronary heart diseases (CHD) in France, despite intake of a high-fat diet, have been attributed to the consumption of red wine containing high levels of polyphenolic compounds. In recent years, understanding the "French Paradox" has stimulated new research interest to investigate whether polyphenolic antioxidants may offer protective effects beyond the cardiovascular system, and whether polyphenols from other botanical sources may similarly offer beneficial effects to human health. Our studies with animal models have provided information clearly indicating the ability of grape polyphenols to ameliorate neuronal damages due to chronic ethanol consumption. Studies with resveratrol, an important component of grape polyphenols, also show protective effects on neuron cell death induced by ethanol and other oxidative agents. These studies demonstrate an urgent need to extend research beyond the "French Paradox" towards better understanding molecular mechanisms of action of polyphenolic compounds and their application to human health.     

Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.     

Differential inhibition of human cytochrome P450 enzymes by epsilon-viniferin,the dimer of resveratrol: comparison with resveratrol and polyphenols from alcoholized beverages

epsilon-Viniferin, a dimer of resveratrol, was isolated in wine at concentration between 0.5 and 5 microM. As resveratrol and polyphenols from red wine were reported to inhibit cytochrome P450 (CYP) activities, this led us to investigate the inhibitory effects of epsilon-viniferin on human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A activities. These effects were compared to those of resveratrol and non volatiles compounds from red wine or various Cognac(R) beverages (enriched with oak-polyphenols). Assays were carried out on human liver microsomes and heterologously expressed CYPs. Ethoxyresorufin, coumarin, benzoxyresorufin, chlorzoxazone, testosterone and lauric acid were used as selective substrates for CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2E1, CYP3A4 and CYP4A, respectively. epsilon-viniferin displayed a more potent inhibitory effect than resveratrol for all the CYP activities tested (Ki 0.5 to 20 microM vs. 10 to 100 microM, respectively). This effect was not due to an inhibition of the NADPH reductase. A particularly potent inhibitory effect was shown for CYP1A1, CYP1B1 and CYP2B6 which are involved in bioactivation of numerous carcinogens. epsilon-viniferin was not a mechanism-based inhibitor of human CYPs. It displayed, like resveratrol, mixed-type inhibitions for all the CYP tested, except for CYP2E1 (non-competitive). Comparison of the inhibitory effects exerted on CYP activities by epsilon-viniferin, resveratrol and non volatile components from red wine or various Cognac beverages showed that neither resveratrol, nor epsilon-viniferin is the main CYP inhibitor present in red wine solids.     

α-Glucosidase inhibitory effect of resveratrol and piceatannol

Dietary polyphenols have been shown to inhibit α-glucosidase, an enzyme target of some antidiabetic drugs. Resveratrol, a polyphenol found in grapes and wine, has been reported to inhibit the activity of yeast α-glucosidase. This triggered our interest to synthesize analogs and determine their effect on mammalian α-glucosidase activity. Using either sucrose or maltose as substrate resveratrol, piceatannol and 3′-hydroxypterostilbene showed strong inhibition of mammalian α-glucosidase activity; pinostilbene, cis-desoxyrhapontigenin and trans-desoxyrhapontigenin had moderate inhibition. Compared to acarbose (IC₅₀ 3–13 μg/ml), piceatannol and resveratrol inhibited mammalian α-glucosidase to a lesser extent (IC₅₀ 14–84 and 111–120 μg/ml, respectively). 3′-Hydroxypterostilbene (IC₅₀ 105–302 μg/ml) was 23–35-fold less potent than acarbose. We investigated the effect of piceatannol and resveratrol on postprandial blood glucose response in high-fat-fed C57Bl/6 mice. Animals administered resveratrol (30 mg/kg body weight [BW]) or piceatannol (14 mg/kg BW) 60 min prior to sucrose or starch loading had a delayed absorption of carbohydrates, resulting in significant lowering of postprandial blood glucose concentrations, similar to the antidiabetic drug acarbose, while no significant effect was observed with the glucose-loaded animals. Our studies demonstrate that the dietary polyphenols resveratrol and piceatannol lower postprandial hyperglycemia and indicate that inhibition of intestinal α-glucosidase activity may be a potential mechanism contributing to their antidiabetic property.     

Estrogen Receptor Alpha as a Key Target of Red Wine Polyphenols Action on the Endothelium

Background

A greater reduction in cardiovascular risk and vascular protection associated with diet rich in polyphenols are generally accepted; however, the molecular targets for polyphenols effects remain unknown. Meanwhile evidences in the literature have enlightened, not only structural similarities between estrogens and polyphenols known as phytoestrogens, but also in their vascular effects. We hypothesized that alpha isoform of estrogen receptor (ERα) could be involved in the transduction of the vascular benefits of polyphenols.

Methodology/Principal Findings

Here, we used ERα deficient mice to show that endothelium-dependent vasorelaxation induced either by red wine polyphenol extract, Provinols™, or delphinidin, an anthocyanin that possesses similar pharmacological profile, is mediated by ERα. Indeed, Provinols™, delphinidin and ERα agonists, 17-beta-estradiol and PPT, are able to induce endothelial vasodilatation in aorta from ERα Wild-Type but not from Knock-Out mice, by activation of nitric oxide (NO) pathway in endothelial cells. Besides, silencing the effects of ERα completely prevented the effects of Provinols™ and delphinidin to activate NO pathway (Src, ERK 1/2, eNOS, caveolin-1) leading to NO production. Furthermore, direct interaction between delphinidin and ERα activator site is demonstrated using both binding assay and docking. Most interestingly, the ability of short term oral administration of Provinols™ to decrease response to serotonin and to enhance sensitivity of the endothelium-dependent relaxation to acetylcholine, associated with concomitant increased NO production and decreased superoxide anions, was completely blunted in ERα deficient mice.

Conclusions/Significance

This study provides evidence that red wine polyphenols, especially delphinidin, exert their endothelial benefits via ERα activation. It is a major breakthrough bringing new insights of the potential therapeutic of polyphenols against cardiovascular pathologies.     

Mechanism of potentiation by polyphenols of contraction in human vein-engineered media

The potential of natural dietary polyphenols in the treatment of vascular diseases originating from veins has been suggested in the literature. However, the mechanisms involved to explain the effects of polyphenols are not yet elucidated. Therefore, the aim of this study was to investigate the mechanisms by which polyphenols from red wine (Provinols) modulated contraction in human veins. We took advantage of a human model previously reported as a new tool for pharmacological research, using tissue-engineered techniques allowing the production of vascular media based exclusively on human smooth muscle cells. Thus human tissue-engineered vascular media (TEVM) were produced with cells originating from umbilical cord vein. TEVM were treated with either vehicle or Provinols. Results showed that treatment of TEVM with Provinols significantly potentiated the contractile responses induced by histamine and bradykinin. The potentiating effect of Provinols was not associated with an enhancement of histamine-induced increase in cytosolic calcium; rather, it implied the presence of a Ca(2+)-independent signaling pathway. Pharmacological studies indicated that action of Provinols took place at the level of phospholipase A(2)-Rho-kinase pathway and was associated with an enhancement of myosin light chain kinase activity. These results, obtained using the human TEVM, bring new insights to explain the regulation of venous contraction by polyphenols.     

Endothelial function and cardiovascular disease: effects of quercetin and wine polyphenols

Endothelial dysfunction is an early pathophysiological feature and independent predictor of poor prognosis in most forms of cardiovascular diseases. Epidemiological studies report an inverse association between dietary flavonoid consumption and mortality from cardiovascular diseases. In the present paper, we review the effects of flavonoids, especially quercetin and wine polyphenols, on endothelial function and dysfunction and its potential protective role in hypertension, ischemic heart disease and stroke. In vitro studies show that flavonoids may exert multiple actions on the NO-guanylyl cyclase pathway, endothelium-derived hyperpolarizing factor(s) and endothelin-1 and protect endothelial cells against apoptosis. In vivo, flavonoids prevent endothelial dysfunction and reduce blood pressure, oxidative stress and end-organ damage in hypertensive animals. Moreover, some clinical studies have shown that flavonoid-rich foods can improve endothelial function in patients with hypertension and ischemic heart disease. Altogether, the available evidence indicates that quercetin and wine polyphenols might be of therapeutic benefit in cardiovascular diseases even though prospective controlled clinical studies are still lacking.     

Antioxidant effect of red wine polyphenols on red blood cells

The protective effect of red wine polyphenols against hydrogen peroxide (H(2)O(2))-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H(2)O(2), were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system.     

Regulation of proliferation and gene expression in cultured human aortic smooth muscle cells by resveratrol and standardized grape extracts

Epidemiologic studies suggest that low to moderate consumption of red wine is inversely associated with the risk of coronary heart disease; the protection is in part attributed to grape-derived polyphenols, notably trans-resveratrol, present in red wine. It is not clear whether the cardioprotective effects of resveratrol can be reproduced by standardized grape extracts (SGE). In the present studies, we determined, using cultured human aortic smooth muscle cells (HASMC), growth and specific gene responses to resveratrol and SGE provided by the California Table Grape Commission. Suppression of HASMC proliferation by resveratrol was accompanied by a dose-dependent increase in the expression of tumor suppressor gene p53 and heat shock protein HSP27. Using resveratrol affinity chromatography and biochemical fractionation procedures, we showed by immunoblot analysis that treatment of HASMC with resveratrol increased the expression of quinone reductase I and II, and also altered their subcellular distribution. Growth of HASMC was significantly inhibited by 70% ethanolic SGE; however, gene expression patterns in various cellular compartments elicited in response to SGE were substantially different from those observed in resveratrol-treated cells. Further, SGE also differed from resveratrol in not being able to induce relaxation of rat carotid arterial rings. These results indicate that distinct mechanisms are involved in the regulation of HASMC growth and gene expression by SGE and resveratrol.